Expression, Purification, and Characterization of Plasmid Recombinant Protein (pGEM-gbr22) in E.coli BL21(DE3)
Introduction
The study of recombinant proteins serve to be the foundation in analyzing the effects of various drugs. Therefore, it is essential to be able to produce and purify a wide range of proteins for further testing and experimentation. There are many factors to take in consideration when dealing with the production and expression of these recombinant proteins such as what organism to express the protein in and whether the protein should be tagged [2]. It is important to realize that each individual protein requires different factors. To produce such a protein, a researcher must obtain DNA and use a T7 RNA polymerase to express the DNA [1]. A numerous amount of proteins are suitable for expression in E.coli therefore its ability as a host is utilized. It is cost efficient, convenient, and effective to test different strategies in E.coli. The BL21(DE3) strain in E.coli is important in producing a high amount of a certain protein. For eukaryotic proteins, the use of BL21(DE3) derivatives has become important in overcoming the effects of codon bias. The protein is suggested to then be produced with an affinity tag to aid in the purification process. Having good conditions for the bacterial lysate will help cell lysis and the avoid sample contamination with genomic DNA. Purifying the protein involves running the protein through a column that contains a column matrix such as nitriloacetate agarose (Ni-NTA). Lastly, the protein must be characterized to assure the purity of the sample with pure protein. Researchers use gel electrophoresis to thoroughly characterize the protein samples into gels. In this lab experiment, the plasmid protein, pGEM-gbr22 will be overexpressed, purified, and characterized in the BL21(DE3) strain of E.coli. If the plasmid is correctly expressed in the bacterial protein, lysed, purified with an affinity tag, and characterized using gel electrophoresis then the protein sample should acquire a high purity and yield of the protein.
Materials & Methods:
First, the bacterial cells were transformed by adding centrifuged pGEM-gbr22 to the BL21(DE3) strain and SOC media onto a agar plate and then incubated. A single colony from the agar plate was placed into the culture tube with fresh LB and ampicillin and incubated for approximately 8 hours and then again for 16-24 hours to achieve a large culture. A sample of the turbid media was stored in the fridge and the rest was centrifuged with a Allegra X-15 benchtop centrifuge (Beckman Coulter, Inc., Brea, CA) to acquire the protein in the form of a pellet. A sample of a the pellet was suspended in phosphate buffered saline with lysozyme and was stored in the freezer. The sample was thawed and Benzonase (Sigma-Aldrich, St. Louis, MO) was added to the solution. The lysate was filtered with a PES syringe filter (Membrane Solutions, Plano, TX). An Econo column (Bio-Rad, Hercules, CA) was used to run the solution with Ni-NTA resin/buffer. Prepared buffers were then ran through the column. A Nanodrop spectrophotometer (Thermo Scientific, Wilmington, ED) was used to measure the absorbance of the plasmid protein from elution samples. The protein was then characterized. Prior samples were prepared with a 6x gel loading buffer and put through SDS gel electrophoresis. The formed gel was then stained with an imperial protein stain and eventually destained with Nanopure water and the use of an orbital shaker. Whatman filter paper (GE Healthcare, Maidstone, UK) was used to cover the gel. The gel was then dried with a vacuum.
Results:
Figure 1. Fun plate with streaks of saliva of Brandon Nguyen and Michael Tra. Plate was incubated at 37 degrees Celsius for 8 hours. No ampicillin.
Figure 2. Control plate with E.Coli strains (BL21(DE3)) and ampicillin. Plasmid DNA, pGEM-gbr22, was not added. No colonies are visible indicating no contamination. Plate was incubated at 37 degrees Celsius.
Figure 3. Plate with E.Coli strains (BL21(DE3)), pGEM-gbr22, and ampicillin. Visible colonies with faint purple color. Protein, pGEM-gbr22, is immune to antibiotic therefore all colonies are bacteria with the expressed protein. Incubated at 37 degrees Celsius for 8 hours.
Figure 4. Large cultures of E.Coli (BL21(DE3)) that express the plasmid protein, pGEM-gbr22, in LB and ampicillin solution. Incubated in shaking incubator at 37 degrees Celsius overnight.
Figure 5. Pellets formed from centrifugation. Pellet consists of bacteria that express purple protein. Supernatant was removed form conicals. The weight of the top conical is 0.50 g and the second conical is 0.67 g.
Figure 6. Elution 1 consisting of purified protein, pGEM-grb22, along with elution buffer containing 5ml of 250 mM imidazole. Elution 2 consists of remaining protein and with same amount of elution buffer.
Figure 7. Absorbance spectra for sample 1 from Elution 1 at 280 nanometers. Absorbance is 0.44. Beer’s Law, A=ebc, states absorbance equals the product of molar absorptivity extinction coefficient, path length, and concentration. With an absorbance of 0.44, extinction coefficient of 38,850, and path length of 10mm (1 cm) the concentration was calculated to being 1.133E-5 M. Using the molecular weight of brr22, 25,794.2 g/mol, the concentration is equal to 0.292 mg/ml.
Figure 8. Absorbance spectra for sample 2 from Elution 1 at 280 nanometers. Absorbance is 0.43. Beer’s Law, A=ebc, states absorbance equals the product of molar absorptivity extinction coefficient, path length, and concentration. With an absorbance of 0.43, extinction coefficient of 38,850, and path length of 10mm (1 cm) the concentration was calculated to being 1.107E-5 M. Using the molecular weight of brr22, 25,794.2 g/mol, the concentration is equal to 0.286 mg/ml.
Figure 9. Absorbance spectra for sample 2 from Elution 1 at 574 nanometers, maximal wavelength of protein. Absorbance is 0.101. Beer’s Law, A=ebc, states absorbance equals the product of molar absorptivity extinction coefficient, path length, and concentration. With an absorbance of 0.101, extinction coefficient of 118,300, and path length of 1mm (.1 cm) the concentration was calculated to being 8.54E-6 M. Using the molecular weight of brr22, 25,794.2 g/mol, the concentration is equal to 0.2202 mg/ml.
Figure 10. Absorbance spectra for sample 2 from Elution 1 at 574 nanometers, maximal wavelength of protein. Absorbance is 0.099 Beer’s Law, A=ebc, states absorbance equals the product of molar absorptivity extinction coefficient, path length, and concentration. With an absorbance of 0.099, extinction coefficient of 118,300, and path length of 1mm (.1 cm) the concentration was calculated to being 8.40E-6 M. Using the molecular weight of brr22, 25,794.2 g/mol, the concentration is equal to 0.216 mg/ml.
Yield if using 280 nm wavelength: (.292 mg/ml + .286 mg/ml)/2 x 4.75ml = 1.373 mg
Yield if using 574 nm, maximal wavelength, (.202 mg/ml + .216 mg/ml)/2 x 4.75ml = .99275 mg
Figure 11. Gel formed by SDS gel electrophoresis for 25 minutes at 200V. Stained with imperial protein stain. First ladder came from fermentas. Ladders 2-7 were Tom Shin's samples 1-6. Ladders 8-10 were Brandon Nguyen's samples 4-6. Position of the purple band signifies molecular weight in kDa and intensity of the color signifies purity of protein.
Figure 12. Cracked gel after drying process in vacuum.
Figure 13. Molecular weight standards ladder regarding SDS gel electrophoresis.
Discussion:
Lysozyme was used to break down the cell wall of the bacteria in this case BL21(DE3). Once the cell wall was broken down, the pGEB-gbr protein could be isolated and purified. Benzonase is a nuclease that reduces the viscosity by digesting the DNA/RNA in the mixture. Sample 1 contained bacterial culture after 16-24 hours of centrifugation, Sample 2 contained supernatant from the lysate, Sample 3 contains sample of the flow of resin and buffer through waste and the Ni-NTA column, Sample 4 contains protein, Ni-NTA resin with wash buffer, Sample 5 contains protein with elution buffer, and Sample 6 contains leftover protein from resin from sample 5 with elution buffer. Wash buffer consists of 1xPBS with 20 mM imidazole and elution buffer consists of 1xPBS with 250 mM imidazole. The HIS tag system is used for protein purification of E. Coli. bacterial cells and strains that have been harvested by centrifugation and the lysing of the resulting cell pellet . Protein samples from the bacterial cell are further purified through imidazole or elution buffer solution. Overall the purity of the protein was satisfactory. The gel showed a decreasing purity with ascending samples. Also the gel shows a basic trend of decreasing in molecular weight as the sample number increased. Sources of error consisted of cracking the gel during the drying process, improper running through the Ni-NTA column, contamination of solution by microorganisms in the breath, improper preparation of buffer solutions.
Conclusions:
In this lab, the protein pGEB-gbr22 was over expressed, purified, and characterized in the bacterial host BL21(DE3). Difference protein samples obtained at different points in time of the entire 3 part procedure were stored in a fridge and used as a comparison for purity of protein. The molecular weight of the protein was approximated to being 20 kDa and the purity was estimated to being 70%. Also the concentration of protein at the maximal wavelength,574 nm, was less than the concentration at 280 nm, the standard. The next immediate step would be to express, purify, and characterize a protein involved with a disease and observe the effects of ligand binding given from GOLD program.
References:
1. Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C., Protein production and purification. Nat Methods2008, 5(2), 135-46.
Expression, Purification, and Characterization of Plasmid Recombinant Protein (pGEM-gbr22) in E.coli BL21(DE3)
Introduction
The study of recombinant proteins serve to be the foundation in analyzing the effects of various drugs. Therefore, it is essential to be able to produce and purify a wide range of proteins for further testing and experimentation. There are many factors to take in consideration when dealing with the production and expression of these recombinant proteins such as what organism to express the protein in and whether the protein should be tagged [2]. It is important to realize that each individual protein requires different factors. To produce such a protein, a researcher must obtain DNA and use a T7 RNA polymerase to express the DNA [1]. A numerous amount of proteins are suitable for expression in E.coli therefore its ability as a host is utilized. It is cost efficient, convenient, and effective to test different strategies in E.coli. The BL21(DE3) strain in E.coli is important in producing a high amount of a certain protein. For eukaryotic proteins, the use of BL21(DE3) derivatives has become important in overcoming the effects of codon bias. The protein is suggested to then be produced with an affinity tag to aid in the purification process. Having good conditions for the bacterial lysate will help cell lysis and the avoid sample contamination with genomic DNA. Purifying the protein involves running the protein through a column that contains a column matrix such as nitriloacetate agarose (Ni-NTA). Lastly, the protein must be characterized to assure the purity of the sample with pure protein. Researchers use gel electrophoresis to thoroughly characterize the protein samples into gels. In this lab experiment, the plasmid protein, pGEM-gbr22 will be overexpressed, purified, and characterized in the BL21(DE3) strain of E.coli. If the plasmid is correctly expressed in the bacterial protein, lysed, purified with an affinity tag, and characterized using gel electrophoresis then the protein sample should acquire a high purity and yield of the protein.
Materials & Methods:
First, the bacterial cells were transformed by adding centrifuged pGEM-gbr22 to the BL21(DE3) strain and SOC media onto a agar plate and then incubated. A single colony from the agar plate was placed into the culture tube with fresh LB and ampicillin and incubated for approximately 8 hours and then again for 16-24 hours to achieve a large culture. A sample of the turbid media was stored in the fridge and the rest was centrifuged with a Allegra X-15 benchtop centrifuge (Beckman Coulter, Inc., Brea, CA) to acquire the protein in the form of a pellet. A sample of a the pellet was suspended in phosphate buffered saline with lysozyme and was stored in the freezer. The sample was thawed and Benzonase (Sigma-Aldrich, St. Louis, MO) was added to the solution. The lysate was filtered with a PES syringe filter (Membrane Solutions, Plano, TX). An Econo column (Bio-Rad, Hercules, CA) was used to run the solution with Ni-NTA resin/buffer. Prepared buffers were then ran through the column. A Nanodrop spectrophotometer (Thermo Scientific, Wilmington, ED) was used to measure the absorbance of the plasmid protein from elution samples. The protein was then characterized. Prior samples were prepared with a 6x gel loading buffer and put through SDS gel electrophoresis. The formed gel was then stained with an imperial protein stain and eventually destained with Nanopure water and the use of an orbital shaker. Whatman filter paper (GE Healthcare, Maidstone, UK) was used to cover the gel. The gel was then dried with a vacuum.
Results:
Yield if using 280 nm wavelength: (.292 mg/ml + .286 mg/ml)/2 x 4.75ml = 1.373 mg
Yield if using 574 nm, maximal wavelength, (.202 mg/ml + .216 mg/ml)/2 x 4.75ml = .99275 mg
Discussion:
Lysozyme was used to break down the cell wall of the bacteria in this case BL21(DE3). Once the cell wall was broken down, the pGEB-gbr protein could be isolated and purified. Benzonase is a nuclease that reduces the viscosity by digesting the DNA/RNA in the mixture. Sample 1 contained bacterial culture after 16-24 hours of centrifugation, Sample 2 contained supernatant from the lysate, Sample 3 contains sample of the flow of resin and buffer through waste and the Ni-NTA column, Sample 4 contains protein, Ni-NTA resin with wash buffer, Sample 5 contains protein with elution buffer, and Sample 6 contains leftover protein from resin from sample 5 with elution buffer. Wash buffer consists of 1xPBS with 20 mM imidazole and elution buffer consists of 1xPBS with 250 mM imidazole. The HIS tag system is used for protein purification of E. Coli. bacterial cells and strains that have been harvested by centrifugation and the lysing of the resulting cell pellet . Protein samples from the bacterial cell are further purified through imidazole or elution buffer solution. Overall the purity of the protein was satisfactory. The gel showed a decreasing purity with ascending samples. Also the gel shows a basic trend of decreasing in molecular weight as the sample number increased. Sources of error consisted of cracking the gel during the drying process, improper running through the Ni-NTA column, contamination of solution by microorganisms in the breath, improper preparation of buffer solutions.
Conclusions:
In this lab, the protein pGEB-gbr22 was over expressed, purified, and characterized in the bacterial host BL21(DE3). Difference protein samples obtained at different points in time of the entire 3 part procedure were stored in a fridge and used as a comparison for purity of protein. The molecular weight of the protein was approximated to being 20 kDa and the purity was estimated to being 70%. Also the concentration of protein at the maximal wavelength,574 nm, was less than the concentration at 280 nm, the standard. The next immediate step would be to express, purify, and characterize a protein involved with a disease and observe the effects of ligand binding given from GOLD program.
References:
1. Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C., Protein production and purification. Nat Methods 2008, 5(2), 135-46.
2. http://www.embl.de/pepcore/pepcore_services/index.html (accessed Apr 15, 2012).